Acta Neuropathologica (2018) 136:41–56

https://doi.org/10.1007/s00401-018-1868-1

ORIGINAL PAPER

Alzheimer’s disease pathology propagation by exosomes containing

toxic amyloid‑beta oligomers
Maitrayee Sardar Sinha1 · Anna Ansell‑Schultz1 · Livia Civitelli1 · Camilla Hildesjö1 · Max Larsson1 · Lars Lannfelt2,3 ·
Martin Ingelsson2 · Martin Hallbeck1 

Received: 20 December 2017 / Revised: 15 May 2018 / Accepted: 19 May 2018 / Published online: 13 June 2018
© The Author(s) 2018

Abstract
The gradual deterioration of cognitive functions in Alzheimer’s disease is paralleled by a hierarchical progression of amy-
loid-beta and tau brain pathology. Recent findings indicate that toxic oligomers of amyloid-beta may cause propagation of
pathology in a prion-like manner, although the underlying mechanisms are incompletely understood. Here we show that
small extracellular vesicles, exosomes, from Alzheimer patients’ brains contain increased levels of amyloid-beta oligomers
and can act as vehicles for the neuron-to-neuron transfer of such toxic species in recipient neurons in culture. Moreover,
blocking the formation, secretion or uptake of exosomes was found to reduce both the spread of oligomers and the related
toxicity. Taken together, our results imply that exosomes are centrally involved in Alzheimer’s disease and that they could
serve as targets for development of new diagnostic and therapeutic principles.

Keywords  Alzheimer’s disease · Exosomes · Oligomers · Beta-amyloid · Human · Prion-like · Propagation

Introduction 38–40, 61] as well as spread of tau between neurons in the

Gradual accumulation of toxic amyloid-beta (Aβ) and tau are
believed to be central to Alzheimer´s disease (AD) patho-
genesis. These abnormal deposits typically appear with a
hierarchical spatial distribution, which suggest that the
pathological proteins can propagate between different brain
areas [7, 52, 53]. Accordingly, recent studies have demon-
strated transfer of Aβ in cellular and animal models [27,
Maitrayee Sardar Sinha and Anna Ansell-Schultz are equal
contributors.
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s0040​1-018-1868-1) contains
supplementary material, which is available to authorized users.
* Martin Hallbeck
Martin.Hallbeck@liu.se
1
Department of Pathology, Department of Clinical
and Experimental Medicine, Linköping University,
Linköping, Sweden
2
Department of Public Health and Caring Sciences,
Geriatrics, Uppsala University, Uppsala, Sweden
3
BioArctic AB, Warfvinges väg 25, 112 85 Stockholm,
Sweden
brain of transgenic mouse models [25]. These observations
are further supported by the distribution of Aß and tau PET
ligands in relation to the brain connectome [45].
Different Aβ conformational states have different proper-
ties, and intermediate products of fibril formation, such as
lower molecular weight Aβ oligomers (oAβ) and protofi-
brils, have been suggested to be particularly neurotoxic and
to act as seeds for further aggregation [27, 61]. In addition,
these soluble forms of Aß correlate better than fibrils with
cognitive function in the AD brain [39]. We have previ-
ously shown that oAβ can accumulate inside cells and sub-
sequently spread from one cell to another [15, 40]. These
findings thus suggest that Aβ can propagate pathology in
a manner similar to what is seen for prion disorders and
for several other neurodegenerative diseases [8]. However,
the underlying mechanisms for the spreading of toxic oAβ
remain incompletely understood [28].
Exosomes, small extracellular vesicles (20–120 nm in diam-
eter) [58] developing from endosomes through multivesicular
bodies, have recently emerged as key players in cellular com-
munication and transport of molecules in both health and dis-
ease, including neuronal toxicity [33] and neurodegenerative
disorders [59]. Exosomes can carry different cargos such as
proteins, RNA and miRNA and can also contain monomeric

13
Vol.:(0123456789)

42 Acta Neuropathologica (2018) 136:41–56

Aß, tau and α-synuclein [12, 17, 46, 59] and can propagate tau
pathology [4]. Thus, exosomes could potentially also carry
aggregated proteins such as oAß. However, no study has so far
investigated if exosomes isolated from AD brain tissue can be
responsible for interneuronal protein transfer.
For the first time, we now demonstrate that intracellular
oAβ is co-localized with exosomes and show that AD brain-
derived exosomes can mediate neuron-to-neuron propaga-
tion of oAß. Furthermore, we show that the concentration of
oAβ in exosomes is significantly increased in post mortem
AD brains. In addition, exosomes carrying oAβ can be inter-
nalized in cultured neurons and spread their toxic content to
nearby cells.
Methods
Brain tissues
Post  mortem brain samples of temporal neocortex from
healthy control (1 female, 4 male) and AD (4 female, 1 male)
subjects (Table 1) were provided by the brain bank at Uppsala
University, Sweden. The tissue was received as fresh-frozen
or as formalin‐fixed (4% formaldehyde) paraffin-embedded
blocks. The AD cases were neuropathologically diagnosed
as CERAD C, Braak stages IV–VI. None of the control
patients suffered from dementia or any neurodegenerative
disorder. The collection and use of post mortem brain tissue
was approved by the Regional Ethical Committee in Uppsala,
Sweden (2005/103, 2005-06-29; 2009/089; 2009-04-22).
Immunohistochemistry and immunofluorescence
of brain sections
Formalin-fixed, paraffin-embedded 10 μm sections of tempo-
ral neocortex from healthy control and AD brains (Table 1)
were used for this study and pretreated as previously
described [6]. After 5 min blocking of endogenous peroxi-
dase by incubation in Background Sniper (Biocare Medical),
slides were washed in Tris-buffered saline (TBS) solution
and incubated with rabbit polyclonal anti-flotillin-1 (Abcam,
1:200) followed by incubation with either the second pri-
mary mouse monoclonal antibody (mAb158, 1:7500, Bio-
Arctic) or mouse monoclonal antibody 82E1 (1:25, IBL
International) for 30 min at room temperature (RT).
After rinsing in TBS, the slides were incubated for 30 min
with MACH 2 Double Stain (Biocare Medical). Following
this step, a 30 min incubation was performed with an AP
linked chromogen IP Warp red/HRP linked chromogen Vina
Green cocktail (Biocare Medical). After rinsing in deionized
water, the sections were counterstained with Mayers haema-
toxylin (Histolab Products AB) and mounted with Pertex
mounting media (Histolab Products AB) and micrographs
were obtained using 100 × oil immersion objective (Nikon
Eclipse 80i, Digital Sight DS-Fi1).
After blocking and incubating with primary antibod-
ies as described above, the slides were rinsed in TBS and
incubated with a Fluorescence Enhancement Probe Mouse/
Fluorescence Enhancement Probe Rabbit cocktail (Biocare
Medical) for 20 min subsequent to a 40 min incubation with
a Goat-anti-Mouse DyLight 549/Goat Anti-Rabbit DyLight
488 cocktail (Biocare Medical) diluted 1:200, respectively.
After rinsing in deionized water, the sections were nuclear
counterstained with DAPI and mounted with Fluoro Care
Anti-Fade Mountant (Biocare Medical) and analysed with a
Zeiss LSM 700 confocal microscope. The differential inter-
face contrast (DIC) mode and 405, 488, 555 and 639 lasers
were used to acquire the images with 63x/1.40 oil immersion
plan-apochromatic DICII objectives. The micrographs were
processed using Huygens (Scientific Volume Imaging) and
ZEN lite (blue edition) software.

Table 1  Demographic and Diagnosis Age Sex PMI Braak Thal CERAD NIA-Reagan αSyn
clinical characteristics of
post mortem cases used in the AD 61 F 48 5–6 5 C High prob None
study
AD 64 M 12 5–6 5 C High prob None
AD 85 F 21 5–6 3 B High prob None
AD 90 F 11 5 3 B None
AD 63 F 48 6 5 C None
C 88 M 39 0 0 0 None
C 88 F 22 2 0 0 In S Nigra
C 63 M 30 0 0 0 None
C 90 M 30 0 1 0 None
C 91 M 27 3 4 C None

5 severe Alzheimer’s disease cases  (Braak stages V–VI) and 5 nondemented cases  (Braak stages 0–III)
were used to determine the presence of oAβ in brain derived exosomes

13
Acta Neuropathologica (2018) 136:41–56 43
Cell lines and differentiation
Two different cultured cell types were used in the study: the
human-induced pluripotent stem cells, AF22 (from a control
subject) and the human neuroblastoma cell line, SH-SY5Y
(ECACC: Sigma-Aldrich). The neuroepithelial stem cell
line, AF22, derived from human-induced pluripotent stem
cells from human skin fibroblasts was provided by Dr. Anna
Falk, Karolinska Institute, Sweden. The process of repro-
gramming human cells was approved by the Ethical Com-
mittee at Karolinska Institute, Sweden (dnr 2012/208-31/3
with addendum 2012/856-32). All samples were given with
informed consent. The AF22 cell line has previously been
shown to have a stable neuronal differentiation competence
and the capacity to generate functionally mature human
neurons [20], denoted hiPSC. The hiPSCs were cultured
on 0.01% poly-l-ornithine and laminin (10 µg/mL, Sigma-
Aldrich) coated cell culture flasks (Corning) in DMEM/
F12 media (Gibco by Life Technologies), supplemented
with EGF (10 ng/mL, PeproTech), FGF2 (10 ng/mL, Pep-
roTech), N2 (5 µl/ml, Life Technologies) and B27 (1 ml/L,
Life Technologies) and further differentiated for 40 days to
functionally mature human neurons in 1:1 DMEM, neuroba-
sal media containing B27 (10 µl/ml), Laminin (1 µl/ml) and
N2 (5 µl/ml) as previously described [20].
Neuronal differentiation of the human neuroblastoma cell
line SH-SY5Y was performed as previously described [1].
In brief, SH-SY5Y cells were cultured and pre-differentiated
for 7 days using 10 μM retinoic acid (RA; Sigma-Aldrich;
denoted as raSH-SY5Y). Pre-differentiated raSH-SY5Y
cells were seeded on 6-, 12- or 24-well glass plates coated
with 20% extracellular matrix (ECM) gel (BD Bioscience)
and further differentiated for 10 days with serum-free MEM
(Gibco by Life Technologies) supplemented with brain-
derived neurotrophic factor (BDNF, 50 ng/ml, PeproTech),
neuregulin β1 (NRGβ1,10 ng/ml, R&D Systems), nerve
growth factor (NGF, 10 ng/ml, R&D Systems) and vitamin
D3 (VitD3, 24 nM, Sigma-Aldrich). These fully differenti-
ated cells are denoted dSH-SY5Y.
In addition, SH-SY5Y cells expressing a CD63-EGFP
fusion protein were generated using AddGene plasmid
#62964.
Labelling and oligomerization of Aβ1‑42
Recombinant Aβ1-42 peptides (Innovagen,) was dissolved
in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma-Aldrich)
and vacuum dried overnight. Aβ1-42 (1.054 mM final con-
centration) was resuspended in N ­ a2CO3 (0.1 M pH 8.5) and
incubated with the fluorophore Alexa Fluor 700 (AF700)
succinimidyl ester (1.58 mM final concentration, Life Tech-
nologies) for 40 min at 4 °C or the fluorophore 6-carboxyte-
tramethylrhodamine succinimidyl ester (TMR, Invitrogen),
in a molar ratio of 2:, incubated overnight at 4 °C. Labelled
Aβ1-42 was diluted to a final concentration of 100 µM in
HEPES 20 mM pH 7.4, vortexed, sonicated for 10 min and
incubated at 4 °C overnight. After the overnight incubation,
Aβ1-42 was separated from free dye with size exclusion
chromatography (SEC). A Sephadex 75 10/300 GL column
coupled to a liquid chromatography system (ÄKTA pure,
GE Healthcare) was equilibrated with ­NH4HCO3 50 mM
pH 8.5 and 500 µl of sample was injected into the column.
To estimate the molecular weight of the Aβ species, LMW
gel filtration calibration kits (GE Healthcare) were used.
Oligomeric and monomeric Aβ species were eluted at a
flow rate of 0.5 ml/min, collected and lyophilized. Then,
Aβ species (oAβ-AF700) were resuspended in phosphate-
buffered saline (PBS) solution and quantified spectrophoto-
metrically at 215 nm by using the Aβ1-42 extinction coef-
ficient (Aβ1-42 ɛ214 nm = 75,887 M−1 cm−1) according to
Lambert–Beer’s law. Protein aliquots were stored at − 80 °C.
Exosome purification, characterization and labelling
Isolation of brain exosomes from extracellular space of
freshly frozen human brain tissues (250 mg) was performed
as previously described [42]. Tissue was dissociated with
papain (20 units/ml, 15 min at 37 °C, Sigma-Aldrich) fol-
lowed by filtration through 40 µm mesh filter (BD Bio-
sciences) and a 0.2 µm syringe filter (Thermo Scientific) to
separate extracellular matrix from cells. The crude exosomes
were then isolated by differential centrifugation method and
subsequently purified by sucrose density gradient as previ-
ously described and resuspended in PBS, lysis buffer or dilu-
ent C (Sigma-Aldrich) for further experiments [42].
Exosomes from conditioned media of raSH-SY5Y cells
were isolated by differential ultracentrifugation. In brief,
50–80 million raSH-SY5Y cells were incubated with oAβ-
AF700for 3 h at 37 °C. After PBS washing, cells were kept
for 48 h in MEM supplemented with exosome-free serum
(System Biosciences). Culture supernatants were collected
and spun at 1000 × g for 10 min for removal of cellular
debris. The supernatants were then filtered through 0.22 µM
filter and sequentially centrifuged at 5000×g, 10,000×g, and
100,000×g. The final pellet was then resuspended in PBS,
lysis buffer or diluent C for further analysis.
Exosomes were labelled with PKH67 or PKH26 dye
(Sigma-Aldrich), according to the manufacturer’s protocol.
Briefly, 4 μL PKH67 dye was mixed with exosome suspen-
sion in diluent C and incubated for 10 min at 37 °C. The
labelling reaction was stopped by adding 20 ml chilled PBS.
Labelled exosomes were ultra-centrifuged at 100,000×g
for 70 min, washed with PBS, ultra-centrifuged again at
100,000×g and the pellet was resuspended in PBS.
13

44
Cellular uptake of exosomes
dSH-SY5Y or hiPSCs cells were plated on coverslips in the
respective serum-free growth medium. Before the uptake
assay, exosomes were isolated from brain tissue or condi-
tioned media of raSH-SY5Y cells and labelled with PKH67
or PKH26 as described above. In order to use equal amounts
of exosomes in the cell cultures, exosomal protein content
was quantitated by using BCA (Bio-Rad) or QuantIT (Inv-
itrogen). Brain exosome abundance was quantified accord-
ing to the AChE activity (EXOCET Exosome Quantification
kit; System Biosciences) according to the manufacturer’s
protocol. The uptake was performed by incubating cell cul-
tures with 100 µl of exosome solutions (corresponding to an
exosomal protein content of 0.62 ± 0.28 μg/μl from brain or
0.71 ± 0.33 μg/μl from conditioned media; equal to 1.4e10
exosome abundance from brain) in a humid chamber for 3 h
(37 °C, 5% C­ O2). For inhibition experiments, cultured cells
were pre-incubated for 30 min with the endocytosis inhibi-
tors, dynasore (dynamin inhibitor, 80 µM), phenylarsine
oxide (clathrin inhibitor 20 µM), genistein (caveolae inhibi-
tor, 200 µM) all from Sigma-Aldrich. Isolated exosomes in
PBS were added to cells for 3 h as above and flow cytometry
was performed.
Co‑culture model
Co-culture of donor-recipient cells was performed by using
two different methods namely the coverslip system (where
physical contact of synapses is possible) or the transwell
system (where physical contact of synapses is not possible).
In both cases, donor cells (raSH-SY5Y 12,500 cells/cm2,
or hiPSCs 25,000 cells/cm2) were seeded on glass cover-
slips coated with 0.1 mg/ml poly-l-ornithine and 10 µg/ml
laminin and cultured as described above for 3 h at 37 °C
with either 1 µM of oAβ-AF700 or labelled exosomes from
brain tissue or conditioned media, and thereafter washed
twice with PBS.
In the transwell system, donor cells were seeded on a
polycarbonate membrane filter with a 0.4 µm pore size (Fal-
con, Corning), placed on top of recipient cells (dSH-SY5Y)
and subsequently co-cultured for 24 h. At the end of incuba-
tion, the membrane filter was removed and recipient cells
were washed with PBS and analysed with flow cytometry or
fixed with 4% PFA for immunofluorescent labelling.
In the coverslip system, the donor cells were seeded on
glass coverslips (VWR International) and placed upside
down on top of recipient cells, predifferentiated as described
above (resulting in donor cells facing recipient cells) and
subsequently co-cultured for 24 or 48 h at 37 °C. For gel
cultured cells this results in a 3D environment. Thereafter,
the coverslips with donor cells were removed and recipi-
ent cells were washed with PBS and either analysed with
13
Acta Neuropathologica (2018) 136:41–56
flow cytometry or fixed with 4% PFA for immunofluorescent
staining. Additionally, to control for donor cell contamina-
tion in the recipient cell samples, donor cells were trans-
fected with BacMam 2.0 early endosomes Rab5a-RFP (Life
Technologies) at a final concentration of 30 particles per cell
before co-culture and RFP fluorescence was monitored in
recipient cells by flow cytometry.
Immunocytochemistry
Co-localization of oAβ and flotillin-1 and TSG101 were
visualized with immunostaining using 1: 5000 solution of
mouse anti-mAb158, 1: 200 solution of mouse anti-flotil-
lin-1 and a 1: 200 solution of mouse anti-TSG101. The sec-
ondary antibodies were Cy3 conjugated (Jackson Immuno
Research, 1:1000) and Alexa Fluor 488-conjugated (Invit-
rogen, 1:400) goat anti-mouse IgG. GFP was detected using
1:200 rabbit anti-GFP (Life Technologies) and 1:400 goat
anti rabbit Alexa flour 647 (Life Technologies).
Cell microscopy
Images of fixed cells were acquired with a Zeiss LSM 700
confocal microscope. The differential interface contrast
(DIC) mode and 405, 488, 555 and 639 lasers were used
to acquire the images with 63x/1.40 oil immersion plan-
apochromatic DICII objectives. Live cell imaging were done
using a Zeiss Primo Vert microscope. The micrographs were
processed using Huygens (Scientific Volume Imaging) and
ZEN lite (blue edition) software.
Flow cytometry
To detect PKH67 labelled exosomes or oAβ-AF700, cells
were released from the ECM gel using Corning Recovery
Solution (Corning) according to manufacturer’s instruc-
tions, filtered through CellTrics 30 µm filters (Sysmex),
re-suspended in PBS, and subsequently analysed on a BD
FACSAria™ (BD Biosciences) flow cytometer.
After inhibiting the gene expression of exosome mark-
ers TSG101 and VPS4A in raSH-SY5Y cells, using RNA
interference, the number of secreted exosomes were ana-
lysed using the Exo-Flow™ kit (System Biosciences, USA),
targeting CD9, CD63 and CD81, as per manufacture’s
instruction.
Enzyme‑linked immunosorbent assay (ELISA)
analysis
Altogether, 96-well EIA/RIA plates (Corning Inc.) were
coated at 4 °C overnight with 200 ng/well of mAb158 in
PBS. Plates were blocked with 1% bovine serum albumin
(BSA) in TBS. Exosome samples prepared in RIPA buffer
Acta Neuropathologica (2018) 136:41–56 45
(150 mM sodium chloride, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate and 50 mM Tris,
pH 8.0) were added to the plates in duplicates and incu-
bated for 2 h at 37 °C. A total of 1 mg/mL of biotinylated
mAb158 was added and incubated for 1 h at 22 °C, followed
by 1 h at RT incubation of streptavidin-coupled poly-HRP
(Mabtech). K-blue enhanced (ANL product, Sweden) was
used as HRP substrate and plates were read in a spectropho-
tometer at 450 nm, using Spectra MAX 190 and then ana-
lysed with SOFT Max Pro. Wells were washed three times
in TBST between each step. The 82E1 sandwich ELISA
was performed same as above using 0.25 µg/ml for capture
and detection antibody [56]. The amount of oAβ within
exosomes was quantified with respect to a standard curve
created with serial dilution of synthetic Aβ oligomers and
expressed as picomolar/mg of protein.
Immunoblot analysis
Exosomes were prepared as described above. Brain lysates
were prepared from homogenised brain tissue followed by
addition of lysis buffer (150 mM NaCl, 0.5% deoxycho-
late, 1% Triton X-100, 50  mM tris-HCL pH 7.5, 20  μl/
ml phosSTOP (Roche), 10 μl/ml Halt Protease inhibitor
cocktail (Thermo Fisher Scientific)), clarified by centrifu-
gation at 10,000 x g for 5 min and sonicated using an ultra-
sonic probe. Cell lysates were prepared from cells collected
in lysis buffer followed by  homogenisation and  sonica-
tion. Samples were mixed with 4x Laemlli loading buffer
and separated on a ClearPAGE SDS Gel 4–12% or 10%
(C.B.S. Scientific), and transferred onto a nitrocellulose
membrane (Invitrogen). The gel was subsequently stained
using InstantBlue protein stain (Expedeon). Additionally,
exosomes, isolated from Control and AD brains or condi-
tioned media of oAβ-AF700-treated raH-SY5Y cells were
lysed by freeze-thawing and subsequently run by SEC using
the conditions described above. The eluted proteins were
collected in fractions of 1 ml, lyophilized, resuspended in
15 µl PBS and spotted on 0.2 µm nitrocellulose membrane.
Membranes were then blocked by 3% BSA followed by pri-
mary antibody incubation. The following antibodies were
used: anti-flotillin-1 (1: 500, BD Transduction Laboratories);
anti-alix (1: 1000, EMD Millipore); anti-TSG101 (1:1000,
Thermo Fisher Scientific); 1:1000, anti-VPS4A (Abcam),
anti-calnexin (1:1000, Abcam), anti-synaptophysin (1:1000,
Synaptic Systems), mAb158 (1: 5000/10000, BioArctic) and
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH,
1:40000, Synaptic Systems,). Anti-rabbit IgG, horseradish
peroxidase (HRP)-linked antibody (1:3000, Dako) and anti-
mouse IgG, HRP-linked antibody (1:3000, Dako) were used
as secondary antibodies. The blots were visualized using
Amersham™ ECL™ (GE Health Care) or S ­ uperSignal®
(Thermo Scientific) detection systems and analysed by
ImageJ software.
Negative staining and transmission electron
microscopy of exosomes
Exosome suspensions were fixed in 4% paraformaldehyde
(at 1:1 dilution, for a final paraformaldehyde concentration
of 2%) overnight at 4 °C and stored at − 20 °C until use.
Thawed exosome suspensions were vortexed briefly and
centrifuged in a microcentrifuge for 30 s. Exosomes were
adsorbed on Formvar-coated Ni mesh grids by placing the
grids on 5 µl drops of exosome suspension for 20 min in a
dry chamber. Negative staining was performed by gently
dripping 100 µl 2% aqueous uranyl acetate onto the grid,
followed by removal of excess uranyl acetate solution using
a lens paper. The grids were examined in a JEOL JEM-1230
electron microscope at 100 kV accelerating voltage. Electron
micrographs were obtained at 150,000–200,000 × magnifica-
tion, for a final image scale of 3.1–4.2 pixels/nm.
Tunable resistive pulse sensing by qNano
Exosome size and particle number were analysed by TRPS
analysis using a qNano instrument (IZON Science, UK) as
described previously [37]. First, isolated exosomes from
brain tissue or conditioned media of dSH-SY5Y cells were
diluted and passed through a 0.2 μm filter (Millipore). Sub-
sequently, particle numbers were counted for a maximum of
5 min or until 500 particles had been counted, using 8 mbar
pressure and the NP150 or NP100 nanopore membranes with
a stretch between 45 and 47 mm. Voltage was set to 0.1 and
0.25 mV to achieve a stable current. Particle size histograms
were recorded when root mean square noise was below 13
pA and particle rate in time was linear. Calibration was per-
formed using known concentration of beads CPC70D (mode
diameter 70 nm) or CPC100B (mode diameter: 110 nm) (all
from IZON) diluted in 1:500 0.2 μm filtered PBS.
Proteinase K digestion
To examine whether exosome-associated Aβ is luminal
or bound to the exterior exosome surface, exosomes were
isolated from conditioned media (oAβAF700-treated) of
dSH-SY5Y cells and incubated with proteinase K (Sigma-
Aldrich,1 mg/ml) for 30 min at 37 °C. 4-(2-aminoethyl)-
benzene-sulfonyl fluoride (Sigma-Aldrich, 0.5 mM) was
subsequently added to the vesicle fraction to inactivate the
enzyme prior to two rounds of 100,000 × g centrifugation.
The final pellet was resuspended in PBS and AF700 fluores-
cence was measured in Tecan Safire2 microplate reader at
Ex/Em 696/719 nm.
13

46
Cytotoxicity assay
To investigate the toxic effect of exosomes on neurons, equal
amounts of exosomes (based on exosomal protein estimation
by BCA) from brains or cells were added to dSH-SY5Y cells
and hiPSCs in our co-culture model for 48 h, as described
above. At the end of incubation, donor cells were removed
and cell medium was collected to assess the release of lactate
dehydrogenase (LDH) in the medium. Collected medium
was centrifuged 2000×g for 5 min at 4 °C and LDH assay
(Pierce) was performed according to manufacturer’s instruc-
tions. The absorbance was measured in a microplate reader
(SpectraMAX 190) at 490 nm with subsequent blank at
680 nm. Furthermore, XTT (2,3-bis [2-methoxy-4-nitro-5-
sulfophenyl]-5-[(phenylamino) carbonyl]-2H-tetrazolium
hydroxide) assay using the Cell Proliferation Kit II (Roche
Diagnostics GmbH) was performed on acceptor cells accord-
ing to the manufacturer’s instructions. The reduced XTT
product produced by mitochondrial enzymes in viable cells,
formazan (bright orange in colour) was measured after 8 h
of incubation at 450 and 750 nm using a Victor 3 V 1420
multilabel plate reader (PerkinElmer). Both LDH and XTT
values were presented as percentage of untreated control.
RNA interference
Cells were seeded at a density of 12 500 cells/cm2 in a
6-well plate and transfected 24 h later with TSG101 or VPS4
mRNA-targeting siRNA or a non-targeting siRNA with no
homology to any known human gene (All Stars Negative
Control siRNA) with the HiPerFect transfection reagent (all
from Qiagen) according to manufacturer’s protocol. TSG#6
(CAGT ​ TTA​ TCA​ TTC​ AAG​ TGT​ AA), TSG#3 (ACTG ​ TCA​ AT​
GTT​ATT​ACT​CTA), VPS#7 (AAG​CTG​AAG​GAT​TAT​TTA​
CGA) and VPS#5 (CTCA ​ AAG​ ACC ​ GAG​ TGA​ CAT
​ AA) siR-
NAs were used for this study. The final siRNA concentra-
tions in the culture medium ranged from 10 to 20 nmol/l.
Twenty-four hours after transfection, knockdown was veri-
fied by quantitative real-time PCR and Western Blot analy-
ses, and a decrease in the mRNA level of 70% or greater was
considered sufficient downregulation.
Gene expression analysis
Total RNA was extracted with the RNeasy Mini Kit (Qia-
gen), and cDNA was obtained with the High Capacity
RNA-to-cDNA Kit (Applied Biosystems). The expression
levels of TSG101 or VPS4 mRNA were analysed with a
7500 Fast Real-Time PCR system and FAM/MGB probes
(Applied Biosystems) to confirm downregulation after
siRNA treatment. All reactions were performed according
13
Acta Neuropathologica (2018) 136:41–56
to the manufacturer’s instructions. GAPDH was amplified as
an internal standard. The data were calculated according to
the comparative Ct method to present the data as fold differ-
ences in the expression levels relative to the control sample.
Statistics
All statistical analyses were performed using GraphPad
Prism Software. Data were expressed as the mean ± SEM,
and statistical comparisons were made using two-tailed
unpaired Student’s t tests with Welch’s correction or one-
way ANOVA with Tukey’s correction. Every batch of cell
cultures was treated as one independent experiment (n = 1).
P values less than or equal to 0.05 were deemed statistically
significant.
Study approval
The collection and use of post mortem brain tissue was
approved by the Regional Ethical Committee in Uppsala,
Sweden (2005/103, 2005-06-29; 2009/089; 2009-04-22).
The process of reprogramming human cells was approved
by the Ethical Committee at Karolinska Institute, Sweden
(dnr 2012/208-31/3 with addendum 2012/856-32). A written
informed consent was received from all donors.
Results
Alzheimer brain exosomes are enriched with oAβ
To examine whether oAβ has the potential to localize to cel-
lular structures that form exosomes in the AD brain we ana-
lysed the co-localization of potential oAβ labelling and the
marker flotillin-1, expressed in multi-vesicular bodies and
exosomes. Brain sections from temporal neocortex from four
post mortem AD brains were double-immunostained with
flotillin-1 and one of two different Aβ antibodies (mAb158
or 82E1). Cells with neuronal morphology displayed both
mAb158 or 82E1 and flotillin-1 labelling (Fig. 1a, b). Next,
the possible co-localization of mAb158 or 82E1 with flotil-
lin-1 was analysed on immunofluorescently labelled, fixed
brain sections using the same antibodies. Although the reso-
lution is not sufficient to prove co-localization, we observed
a strong correlation between mAb158 or 82E1 and flotillin-1
labelling, suggesting a subcellular co-localization (Fig. 1c,
d) (R2 = 0.78 and 0.86 for mAb158 and 82E1, respectively).
The detected fluorescence was not due to lipofuscin-derived
autofluorescence (Supplementary Fig. S1a). Currently, no
antibodies with verified oAβ selectivity in IHC applications
have been reported. Thus, we selected the two Aβ antibod-
ies with most extensive data on oligomer selectivity in other
Acta Neuropathologica (2018) 136:41–56 47

a c e f
Brain lysate Exosome Control AD

mAb158 flotillin-1 DAPI

mAb158 flotillin-1 DAPI

Flotillin-1 Alix

Alix Flotillin-1

Calnexin

Synaptophysin

b d g h
82E1 flotillin-1 DAPI
82E1 flotillin-1 DAPI

pM oAß / mg protein
pM oAß / mg protein
*

Control brain AD brain Control brain AD brain

i j Control brain exo

l
Control cell exo
Absorbance (215 nm)

Absorbance (700 nm)

AD brain exo
oAß cell exo
Pure Aß
Flotillin-1 pg/ml

Time (min) Time (min)

k 12 14 16 18 20 22 24 26 28 min m 12 16 18 20 22 24 26 28 min
Control brain exo Control cell exo
Control brain AD brain
AD brain exo oAß cell exo

Fig. 1  AD brain exosomes are enriched with oAβ. AD brain sections
from temporal neocortex show co-localization of probable oAβ to
exosomes. a, b Double-immunostaining with the exosome marker flo-
tillin-1 (labelled red), and two different oligomer-selective (see main
text) Aβ antibodies mAb158 and 82E1, respectively (labelled blue-
green). Scale bar 10 µm. c, d Co-localization of oAβ to exosomes was
performed after immunofluorescence labelling. mAb158 or 82E1,
respectively, showed substantial co-localization (yellow) with flotil-
lin-1 inside cells in the AD brain (R2 = 0.78 and 0.86 for mAb158 and
82E1, respectively). Scale bar, 5  µm. e Immunoblot showing flotil-
lin-1, alix, calnexin and synaptophysin in exosome and brain lysate,
demonstrating no cellular or synapse vesicle contamination in the
exosome preparation. Loading control is shown in Supplementary
Fig. S1d. f Immunoblot demonstrating the presence of flotillin-1 and
alix, in exosome fractions isolated from control and AD brain. Quan-
titative ELISA analysis of oAβ in exosomes isolated from AD and
control brains, using 82E1 (h) and mAb158 (g) antibodies, respec-
tively. Data are presented as the mean picomolar of oAβ per mg total
exosome protein ± SEM (n = 3). *p < 0.05 by two-tailed unpaired Stu-
dent’s t tests with Welch’s correction. i Quantitative analysis of flo-
tillin-1 by ELISA in control and AD brain exosomes showing equal
amount of flotillin-1 between the groups. j Representative SEC pro-
file of lysed exosomes isolated from control and AD brain samples
at 215  nm absorbance (general protein detection). k Detection of
oAβ in SEC eluate fractions of AD and control brain exosomes by
dot blot using mAb158 antibody. l SEC chromatograms of exosomes
isolated from conditioned media of control- or oAβ-AF700 treated
dSH-SY5Y cells as well as pure oAβ-AF700. Detection at 700  nm
absorbance (AF700 detection). m Dot blot detection of oAβ (mAb158
antibody) in SEC eluate fractions of exosomes isolated from condi-
tioned media of control- or oAβ-AF700 treated dSH-SY5Y cells of
control and oAβ-AF700 treated dSH-SY5Y cells exosomes. The anal-
ysis of SEC fractions confirms the presence of oAβ in exosomes

assays (mainly ELISA). Both these antibodies showed including multivesicular bodies, in the human AD brain,
similar labelling patterns in brain tissue (Fig. 1a–d). Fur- which thus suggests that oAß could be released in exosomes.
thermore, our findings from dot-blot (Fig. 1k, m, described Since oAβ is believed to be central to AD pathophysi-
below) supports that mAb158 has a selectivity for aggre- ology [50], we postulated that exosomes from AD brains
gates over monomers also in assays where the protein is would contain higher concentrations of oAβ when com-
in a bound state. Taken together, these data indicate that pared to similar preparations of control brain samples from
the identified Aβ labelling likely represents oligomeric Aβ patients deceased from non-neurological reasons. Using
aggregates. Thus, our findings suggest an association of well-established methods for mild dissociation of nerv-
oAβ with intracellular compartments containing flotillin-1, ous tissue followed by separation of extracellular matrix
from cells we were able to isolate exosomes by sequential

13

48 Acta Neuropathologica (2018) 136:41–56

Exosomes oAß
a b c

Or

labeling of exosomes
containing oAβ from
AD brain or cells in culture
d e
2. Uptake by donor cells
on glass slides

figure b-c
and
figure f-g

3. Co-culture slides upside down

on top of acceptor cells

f g

4. Co-culture for 48 h

5. Removal of the donor cell slide

Merged
Red

figure d-e
and
figure h

confocal microscopy and cytotoxicity

assays

i j
Exosomes PKH67 Exosome free fraction PKH67 CD63 GFP expressing exosomes Exosomes PKH26

13
 Acta Neuropathologica (2018) 136:41–56 49
◂Fig. 2  Exosome-mediated uptake and propagation of oAβ in neu-
ronal cells. Exosomes isolated from brain tissue or conditioned
media of dSH-SY5Y cells were labelled with the dye PKH67 and
added to donor hiPSCs or dSH-SY5Y cells. After 3  h of incubation
at 37  °C, donor cells were fixed, stained with mAb158 (for brain
exosomes) and analysed by confocal microscopy or donor cells were
co-cultured with another set of hiPSCs or dSH-SY5Y (recipient
cells). After 48 h of co-culture, donor cells were removed and recipi-
ent cells were fixed, stained with mAb158 (for brain exosomes) and
analysed by confocal microscopy. a A cartoon illustrating the co-
culture model with hiPSC or dSH-SY5Y cells employed to measure
the transfer of the brain or cell exosomes containing oAβ. Uptake of
b control and c AD brain exosomes (green) containing oAβ (red) in
hiPSC donor  cells. Transfer of AD brain exosomes (green) contain-
ing oAβ (red) to recipient d hiPSCs and e dSH-SY5Y cells. Uptake
of exosomes (green) containing oAβ-AF700 (red) in donor f dSH-
SY5Y and g hiPSCs. h Transfer of oAβ-AF700 containing exosomes
in recipient dSH-SY5Y cells. Super-imposed image of the red (oAβ)
and green (exosomes) channels on a DIC image shows co-localization
(yellow) of exosomes and oAβ. Arrows indicate exosomes or exo-
some containing oAβ. i Cellular uptake of isolated brain exosomes
and brain exosome free fraction after PKH67 staining showing no
PKH67 uptake in the absence of exosomes. j Isolated exosomes from
CD63-GFP expressing SH-SY5Y cells, double-labelled with PKH26
(red), were added to dSH-SY5Y cells. The CD63-GFP were intensi-
fied using an anti-GFP antibody. Scale bar (b–h), j 20 µm, i 10 µm
ultracentrifugation from fresh-frozen, post mortem brain tis-
sues from temporal neocortex of both AD and control sub-
jects. Analyses of the isolated fractions by Tunable Resis-
tive Pulse Sensing technology (TRPS, qNano system) and
transmission electron microscopy (TEM, Supplementary
Fig. S1b, c) demonstrated presence of vesicles with a size
corresponding to exosomes [55]. Immunoblotting confirmed
the extracellular origin of the extracted vesicles by presence
of the exosomal markers alix and flotillin-1 and the lack of
the cellular marker calnexin and the synapse vesicle marker
synaptophysin (Fig. 1e). Furthermore, immunoblotting con-
firms the presence of exosomal markers in extracted vesi-
cles from both control and AD brains (Fig. 1f). As immu-
nostaining of flotillin-1 and oAβ in the AD brain sections
does not have the sufficient resolution, ELISA, which is
a more quantitative method, was implemented to demon-
strate the oAβ in brain exosomes. When used as capture and
target in an ELISA the Aβ antibodies mAb158 is selective
for Aβ oligomers [19, 57], while 82E1 detects both soluble
and fibril Aβ [31]. Interestingly, ELISA with either one of
these two oligomer-selective antibodies showed significantly
higher levels of oAß in exosomes from AD brains (n = 5,
3.12 ± 0.93 pM oAß/mg protein for 82E1 and 7.69 ± 1.68 pM
oAß/mg protein for mAb158) than in those from healthy
control brains (n = 5, 0.46 ± 0.30 pM oAß/mg protein for
82E1 and 2.66 ± 0.74 pM oAß/mg protein for mAb158)
(Fig.  1g, h). There was  no significant difference in the
amount of flotillin-1 in exosomes derived from AD or con-
trol brain tissue of equivalent weight (Fig. 1i). The increase
in exosomal oAß was not an effect of co-precipitation of
free oAß during the extraction as spiking exosomes with
exogenous oAß did not result in increased levels, which
also implies that association of oAß with exosomes occurs
intracellularly (Supplementary Fig. S1e). Additionally, the
presence of oAβ in isolated exosomes was further assessed
using size exclusion chromatography (SEC). SEC chroma-
tograms of the isolated and lysed exosomes from control
and AD brain samples were analysed at 215 nm absorbance
(general protein detection) and proteins were detected in the
void volume (first peak) as well as in fractions correspond-
ing to lower molecular weight (Fig. 1j). The presence of
oAβ in the different SEC eluate fractions was further inves-
tigated by dot-blot analysis using mAb158. This confirmed
that exosomes isolated from AD brain samples contained
oAβ in SEC fractions running at the oligomer size that
was not detected in control brain (Fig. 1k). Similarly, lysed
exosomes isolated from conditioned media of control- or
oAβ-AF700 treated neuronally  differentiated neuroblas-
toma cells (dSH-SY5Y) as well as pure oAβ-AF700 were
analysed on SEC chromatograms with detection at 700 nm
absorbance (AF700 detection). This shows absorbance
peaks (oligomeric and monomeric) at the same time points
(size) for exosomes from conditioned media of oAβ-AF700
treated cells as for the pure oAβ-AF700 initially used to treat
the cells (Fig. 1l). oAβ was detected only in fractions from
exosomes from conditioned media of oAβ-AF700 treated
dSH-SY5Y cells and not in control cell exosomes (Fig. 1m).
Furthermore, these findings show that, in addition to previ-
ously shown Aβ oligomer selectivity in ELISA assay [19,
57], the mAb158 antibody is oligomer selective also in an
assay where protein is not free floating.
The localization of oAß to exosomes was further inves-
tigated in neuronal cell models. dSH-SY5Y or hiPSC were
incubated with oAβ-AF700 for 3 h, after 48 h exosomes were
isolated from the conditioned media by sequential ultracen-
trifugation. Similar to the brain extractions, the isolated vesi-
cles were characterized as exosomes using immunoblotting
for the exosome markers, alix and flotillin-1, lack of cellular
marker calnexin and synapse vesicle marker synaptophysin,
TRPS and TEM (Supplementary Fig. S2a–d) [35, 54]. To
investigate the location of oAß, exosomes isolated from con-
ditioned media from oAß-AF700 treated retinoic acid dif-
ferentiated SH-SY5Y cells (raSH-SY5Y) were treated with
proteinase K (1 mg/ml) to digest proteins on the surface,
confirming that the localization of oAβ is mainly luminal
as this did not significantly decrease exosomal oAβAF700
(Supplementary Fig. S2e).
Exosomes from AD brain are internalized
and transferred by neurons, causing cytotoxicity
To investigate whether exosomes could be a vehicle for the
spreading of oAß, we investigated if exosomes isolated from
13

50
AD brains could be taken up by hiPSCs and dSH-SY5Y cells
(Fig. 2a). The cells were incubated with exosomes (PKH67
labelled) isolated from brain tissue. After 3 h, both exosomes
(Fig. 2b) and oAß (mAb158, Fig. 2c) had been taken up by
the cells. Using a co-culture system with hiPSCs or dSH-
SY5Y cells, previously shown to result in synaptically con-
nected neurons [40], we next investigated the possible trans-
fer of brain-derived exosomal oAß between these neurons.
When RFP labelled donor cells that had been exposed to
exosomes were placed on top of recipient neurons for 48 h,
a substantial number of transferred exosomes (PKH67) and
oAβ (mAb158) could be detected in both types of recipi-
ent cells (Fig. 2d, e). Moreover, transferred oAß was still
partly co-localized with PKH67 labelled exosomes in the
recipient cells, suggesting that after being internalized part
of the exosomes can be transferred onwards, still intact with
their oAß content. Similarly, uptake of oAß in raSH-SY5Y
treated with oAβ-AF700 (3 h) resulted in a co-localization
with intracellular flotillin-1 and TSG101 (Supplementary
Fig. S3a-b). Isolated (48 h conditioned media) and labelled
(PKH67) exosomes were readily taken up by new cells. A
high degree of internalization of exosomes in either dSH-
SY5Y (Fig. 2f) or hiPSC (Fig. 2g) cells was observed. These
vesicular structures had a mainly perinuclear localization
and many of them were positive for oAβ-AF700. Using
these secondary neurons as donor cells and co-culturing
them with a new set of dSH-SY5Y recipient cells for 24 h
showed further spread of AF700 labelled oAβ to this third
set of cells (red channel, Fig. 2h). Similarly to what we
observed for exosomes from AD brains, we could detect a
substantial fraction of oAβ that was still co-localized with
exosomes (merged, Fig. 2h). These findings support that,
apart from releasing their cargo, intact exosomes also can
carry and transfer oAβ further to new recipient cells. Like-
wise, exosomes isolated from conditioned media from cells
that had not been exposed to oAβ were also taken up by
cells and transferred onwards (Supplementary Fig. S3c),
corroborating that the release and uptake of exosomes per
se is not dependent on the presence of oAß. To confirm that
the observed PKH labelling is not due to artefacts we used
the supernatant from the last ultracentrifugation wash-step
of the exosome isolation from brain or conditioned media,
respectively. The supernatant was labelled with PKH67
using the same protocol as for labelling the exosomes. The
labelled sample were subsequently added to raSH-SY5Y
cells. Contrary to the exosome fraction the exosome-free
fraction did not result in any labelling in the cells either in
brain (Fig. 2i) or conditioned media extracts (Supplemen-
tary Fig. S3d). Furthermore, isolated exosomes from CD63-
GFP expressing raSH-SY5Y cells were double-labelled with
PKH26 (red) and added to raSH-SY5Y cells (Fig. 2j). This
confirmed that the exosomes studied here are not artefacts,
but indeed exosomes.
13
Acta Neuropathologica (2018) 136:41–56
Further evidence for the exosomal transportation of oAβ
was obtained by using a transwell co-culture model after
uptake of oAβ containing exosomes to donor cells. Also,
in this model oAβ transferred to recipient cells, suggesting
vesicle transfer of oAβ between cells without direct neuritic
connections. However, compared to the coverslip co-culture
model significantly less neurons containing transfer of oAβ
was detected in the transwell model, a decrease from 23.60
to 5.18% as analysed by flow cytometry and illustrated by
confocal imaging (Supplementary Fig. S4a, b). Thus, sug-
gesting that transfer is more efficient between cells in prox-
imity, which could potentially depend on multiple mecha-
nisms of transfer.
Next, we investigated if the spread of exosomes and their
oAß cargo could result in neuronal toxicity. Isolated brain
exosomes (equalized by protein amounts) from either control
or AD brains were added to dSH-SY5Y cells for 3 h, these
cells were used as donor cells in 48 h co-culture with recipi-
ent dSH-SY5Y cells. The recipient cells were subsequently
investigated for morphological changes compared to control
co-culture with untreated donor cells (Fig. 3a). As described
previously [2] the control dSH-SY5Y shows neuronal mor-
phology with long, branching networks of neurites. Similar
morphology was seen in recipient cells in the control brain
exosome conditions. Occasional changes in neurite mor-
phology could be identified. Interestingly, more pronounced
pathology was seen in recipient cells co-cultured with AD
brain exosome treated donor cells. Here dystrophic neur-
ites with neurite beading and loss of neurite branching were
identified, known early signs of neurodegeneration [51]. We
quantified these effects further and notably, we found that
the transfer of AD brain exosomes induced significant cyto-
toxicity compared to control brain exosomes, as assessed by
the degree of membrane leakage of lactate dehydrogenase
enzyme (LDH) from the recipient hiPSC and dSH-SY5Y
cells (Fig. 3b, c). Induced cellular toxicity was also detected
by XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-5-
[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) analy-
sis in both cell types (Fig. 3d, e). Similar toxicity was further
seen after treatment with exosomes from conditioned oAß
media (Fig. 5d). In addition, exosomes from control brains
which should contain significantly less oAß, caused toxicity
in hiPSCs but not in dSH-SY5Y cells (Fig. 3b, e). No toxic-
ity was seen after treatment with exosomes from conditioned
media from cells not treated with oAβ (Supplementary Fig.
S5a). Taken together, these results suggest that intact AD
brain exosomes, carrying oAβ, are taken up by neurons and
migrate to second order neurons where they can release their
cargo and cause cytotoxicity.
Acta Neuropathologica (2018) 136:41–56 51
a Control
hiPSC
b c
*
Cell viability LDH (%)
***
-------------------------------
AD brain
d hiPSC
Cell viability XTT (%)
-------------------------------
ns
***
AD brain
Fig. 3  Transfer of AD brain exosomes causes cytotoxicity. Exosomes
isolated from control and AD brain tissues were added to donor hiP-
SCs or dSH-SY5Y cells. After 3 h of incubation at 37 °C, donor cells
were washed with PBS and co-cultured with another set of hiPSCs
or dSH-SY5Y (recipient cells). After 48 h of co-culture, donor cells
were removed. a Morphological changes assessed in recipient dSH-
SY5Y showing loss of neurite branching after transfer of AD brain
exosomes. Also, neurite beading was seen in dystrophic neurites as
Inhibition of exosome formation and secretion
inhibit the spread of oAβ
If exosomes are capable of transferring oAß between neu-
rons, it should be possible to stop this transfer by inhibiting
the formation of exosomes. As previously shown, biogen-
esis of exosomes and its cargo proteins can be modulated
by knocking down two Endosomal Sorting Complexes
Required for Transport (ESCRT) proteins, TSG101 and
VPS4A, required for exosome formation and secretion,
respectively [3, 18, 21, 30]. By using siRNA oligonucleo-
tides, a significant decrease in the mRNA levels (decreased
in average with 90% for TSG101 and 88% for VPS4A),
protein levels (decreased in average with 87% for TSG101
and 65% for VPS4A) and the number of secreted exosomes
(decreased in average with 95% for TSG101 and 76% for
Control brain exo AD brain exo
dSH-SY5Y
*
***
Cell viability LDH (%)
ns
** -------------------------------
Control brain AD brain Control brain
e dSH-SY5Y
ns
Cell viability XTT (%)
ns
-------------------------------
**
**
Control brain AD brain Control brain
shown in magnified insert. The conditioned media was collected for
LDH assay (b, c) and recipient cell viability evaluated by XTT (d, e).
Values are expressed as percentage of untreated control. Values are
mean ± SEM (n = 6 separate experiments). LDH shows that transfer
of AD brain exosomes causes significant higher cytotoxicity com-
pared to control brain exosomes in both cell types. NS, not signifi-
cant; *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed unpaired Stu-
dent’s t tests with Welch’s correction
VPS4A) (Fig. 4a–c) was achieved. The siRNA treatment
did not affect viability, as analyses showed that > 95% of
the cells were viable as measured by XTT assay (Fig. 4d).
To distinguish between different mechanisms of transfer, we
again used the coverslip and transwell co-culture models
and quantified the oAβ transfer in presence of TSG101 or
VPS4A siRNA in our co-culture model using flow-cytome-
try. Interestingly, upon inhibition of exosome formation and
secretion the oAβ transfer was almost completely blocked in
the transwell model, whereas almost half of the oAβ transfer
could be stopped in the coverslip model (Fig. 4e). These
results indicate that exosomes are largely responsible for the
neuron-to-neuron transfer of oAβ, although other mecha-
nisms of transfer may also be involved.
13

52 Acta Neuropathologica (2018) 136:41–56

a b
TSG101 VPS4A
Relative mRNA expression

Relative protein levels

-------------------------------------------

46 kDa 50 kDa
*

*** *** **
GAPDH

TSG101 VPS4A siRNA Neg C TSG101 Neg C VPS4A Neg C TSG101 VPS4A

c d e
Number of exosomes (%)

Cell viability XTT (%)

------------------------------------------ -------------------------------------------

oAß transfer (%)

----------------------------------------- TSG101
*** VPS4A
***
**

*** *** ***

TSG101 VPS4A Neg C TSG101 VPS4A Coverslip Transwell

Fig. 4  Downregulation of exosomal proteins TSG101 and VPS4A toxicity was detected by XTT assay after 48  h of transfection with
inhibits the spread of oAβ. Depletion of TSG101 and VPS4A by siRNA. e Quantification of oAβ transfer in presence of TSG101 or
siRNA in dSH-SY5Y cells. a Real time PCR analysis of mRNA VPS4A siRNA in both coverslip and transwell co-culture model by
expressions to show knock down efficiency of transfected cells by flow cytometry, shows that both siRNAs significantly inhibit oAβ
TSG101 or VPS4A siRNA. b Representative immunoblot picture of transfer. Values are expressed as percentage of siRNA negative con-
cell lysates after siRNA treatment for 72  h and associated densito- trol and indicated as dotted line. Values are the mean ± SEM (n = 4
metric analysis (n = 3). c Bead flow cytometry analysis of exosomes separate cultures), *p < 0.05, **p < 0.01, ***p < 0.001 by two-tailed
shows a significant decrease in the number of secreted exosomes after unpaired Student’s t tests with Welch’s correction
TSG101 or VPS4A siRNA treatment in raSH-SY5Y cells. d No cyto-

The uptake of exosomes and spread of oAß with dynasore treatment, which almost completely abolished
is dynamin‑dependent and can be blocked the uptake of exosomes (Fig. 5a), while having only minor
effect on the internalization of oAβ added directly to the
After being released, the exosomes can be taken up by the medium (Fig. 5b). This observation supports that the uptake
recipient cells via different mechanisms. In dSH-SY5Y of exosomes, and thus also oAß transferred via this route, is
cells the uptake of isolated exosomes observed at 37 °C, as regulated by dynamin.
described above, was completely abrogated at 4 °C (Supple- The dependence on dynamin, and thus exosomes, for
mentary Fig. S5b). Thus, the exosomal uptake is an active the spread of oAß between neurons was further confirmed
process. Neuronal cells are capable of different modes of using dSH-SY5Y cells in the transwell model using donor
endocytosis, ranging from receptor-mediated and clathrin- cells fed with exosomes, isolated from conditioned media
dependent, to independent endocytosis [10, 34, 47]. To (oAβ-AF700 treated raSH-SY5Y cells). In this setting with
explore the uptake mechanism, we treated dSH-SY5Y cells an absence of direct neuritic connections, oAβ transfer was
with isolated, PKH67 labelled, exosomes together with vari- almost completely blocked by dynamin inhibition (Fig. 5c),
ous inhibitors of endocytosis: dynasore (dynamin inhibitor), in line with a major dependence on exosomes for transfer.
phenylarsine oxide (clathrin inhibitor) and genistein (cave- When instead performing the same experiment using the
olae inhibitor). Treatment with phenylarsine oxide caused coverslip co-culture model, allowing for direct cell-to-cell
minor cellular toxicity, while no notable cellular toxicity was contacts, there was also a significant decrease of transferred
observed upon treatment with dynasore or genistein (Sup- oAß (Fig.  5c). Since our observations substantiate the
plementary Fig. S5c). Strikingly, all three inhibitors caused hypothesis that exosomes play a significant role in trans-
a significant decrease in the proportion of cells that took ferring oAβ from one neuron to another, we next sought
up exosomes as quantified by flow-cytometry; the decrease to investigate whether transfer of exosomes containing oAβ
was 97.7% with dynasore, 66.5% with phenylarsine oxide had any direct toxic effect on the recipient cells. Cell-to-
and 76.0% with genistein. Thus, the effect was most marked cell transfer of exosomes, isolated from conditioned media

13
Acta Neuropathologica (2018) 136:41–56 53
a b
---------------------------------------------
Exosome uptake (%)
***
***
Dynasore Phenylarsine
oxide
c d
-----------------------------------------------
oAß transfer (%)
ns
***
Coverslip
Fig. 5  The uptake of exosomes and the subsequent spreading of oAß
is dynamin-dependent. a, b Uptake of PKH67 labelled exosomes
or oAβ-AF700 in dSH-SY5Y cells. Cells were pre-incubated with
the indicated inhibitors for 30  min, then exposed to exosomes or
oAβ-AF700. After 3  h incubation, samples were collected and
the proportion of cells with uptake was quantified by flow cytome-
try and related to untreated control (dotted line). c Flow cytometry
analysis of oAβ transfer in presence of dynasore in coverslip and
transwell co-culture models. After dynasore treatment there is a sig-
(oAβ-AF700 treated raSH-SY5Y cells), showed a significant
increase in cellular toxicity of 26% in the recipient cell, as
evident by LDH assay (Fig. 5d). Exosomes from untreated
raSH-SY5Y cells had no significant toxic effect on the recip-
ient cells (Supplementary Fig. S5a). Importantly, we found
that such induced toxicity could be abolished by blocking the
transfer of exosomes using dynasore (Fig. 5d). These data
provide proof-of-concept that the transfer of exosomes and
oAβ together with subsequent toxicity, can be prevented by
inhibiting the dynamin-dependent uptake pathway.
Discussion
Recent evidence suggests that toxic Aβ aggregates can
spread pathology in the Alzheimer brain. The nature of the
propagating species has not been established, although sev-
eral studies have indicated a particularly pathogenic role of
soluble oAβ on synaptic and cellular functions and structure
[13, 32, 50, 60]. It has been speculated that exosomes might
transfer neurodegenerative proteins in the affected brain [28].
Accordingly, exosomes from blood [22], CSF [17, 49] and
ns
oAß uptake (%)
- - - - - - - - - - - - - - - - - - - - - - - -ns
--------------------------
**
***
Genistein Dynasore Phenylarsine Genistein
oxide
**
**
Cell viability LDH (%)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -ns
-----------
***
Transwell oAß oAß+Dynasore
nificant decrease of the proportion of cells with oAβ transfer in both
models (control, dotted line). d Transfer of exosomes isolated from
oAβ treated cells causes cytotoxicity in recipient cells compared to
untreated control as shown by LDH assay, whereas dynasore treat-
ment significantly reduces the cytotoxic effect versus untreated con-
trol (dotted line). Data are represented as the mean ± SEM, NS, not
significant; n = 4; **p < 0.01, ***p < 0.001 by two-tailed unpaired
Student’s t tests with Welch’s correction
cell cultures [46] have been shown to contain monomeric
Aβ and tau, but so far, no study has addressed the presence
of oAβ in exosomes from human AD brains. In this study,
we could show that AD brain exosomes contain an increased
amount of oAβ compared to non-neurological control brains
and found evidence that exosomes can be responsible for
the neuron-to-neuron transfer of toxic oAβ. These findings
suggest that exosomes might be the main mediator of the
pathogenic progression in AD as was recently suggested for
dementia with Lewy bodies [41]. In addition, we found a
co-localization between oAβ and exosomes inside neurons,
which might indicate that exosomes play a role in Aβ sort-
ing and oligomerization [16]. The AD brain exosomes were
further shown to effectively transfer oAβ from one neuron
to another, with subsequent toxic effects on the recipient
cells. Interestingly, at least a part of the exosomes seems
to be transferred intact to further cells, consistent with a
recent study showing that a substantial fraction of exosomes
internalized in one cell were subsequently passed on to a
second cell [44]. Importantly, there is increasing evidence of
correlations between intra-neuronal oAβ and cell death [43].
We have previously demonstrated that transfer of oAβ causes
13

54
neurotoxicity [40] which has also been shown with Aß con-
taining exosomes isolated from AD CSF [33]. Accordingly,
we now found signs of neurotoxicity both morphologically
and with the LDH and the XTT assays after transfer of
exosomes carrying oAβ. This observation not only rein-
forces the role of intracellular oAβ in AD pathogenesis but
also establishes the disease relevance associated with the
exosomal neuron-to-neuron transfer of intercellular oAβ.
The molecular content of exosomes is a fingerprint of
the releasing cell type and, because of their small size,
neuronal exosomes are released into accessible body fluids
such as blood and CSF [48]. Since neuronal exosomes dis-
play unique neuron-specific surface markers [22, 26] they
may be a valuable biomedical marker for early diagnosis
and treatment in AD. Indeed, exosomes have recently been
highlighted as diagnostic biomarkers in various disease con-
ditions, including AD [29, 36]. In concordance, the finding
of increased levels of oAβ in brain exosomes opens the pos-
sibility that similar features could be detected also in eas-
ily accessible body fluids, such as plasma and CSF. Hence,
measurement of increased oAβ in exosomes from such
patient samples could potentially serve as a diagnostic tool.
The intercellular propagation of oAβ and its ensuing
toxicity could also serve as a potential treatment target by
inhibiting either formation, secretion, or cellular uptake of
exosomes. Indeed, downregulation of TSG101 and VPS4A,
proteins necessary for exosome formation and secretion,
was found to result in decreased release of exosomes and
a reduced subsequent transfer of oAβ, thus supporting the
possibility of modulating this mechanism. Moreover, these
observations are in line with recent studies showing that
interfering with exosome release can impact the release of
specific proteins [9, 14]. An alternative therapeutic target
could be the dynamin-dependent uptake of exosomes [23,
24] as the dynamin inhibitor dynasore decreased exosome
propagation, spread of oAβ and the associated neuronal tox-
icity, leading to rescued cell viability. Dynasore itself would
not be a feasible therapeutic substance, but phenothiazine-
derived antipsychotic drugs have been suggested to inhibit
dynamin dependent endocytosis [11] and could thus be suit-
able for further drug development.
In conclusion, our results point to a role for exosomes
in the spreading of toxic oAβ and the associated disease
progression in the AD brain (summarized in Supplemen-
tary Fig. S6). It has been suggested that exosomal release
may provide an alternative disposal mechanism to lysoso-
mal degradation of oAβ [5] or other proteins that are resist-
ant to degradation [55]. We speculate that this alternative
mechanism of clearance, which initially could be beneficial
for the cells, over time becomes a liability with increased
propagation of pathological proteins throughout the brain.
The possibility of inhibiting exosome transfer and the related
13
Acta Neuropathologica (2018) 136:41–56
spread and toxicity of oAβ may lead to the identification of
new pharmaceutical targets for AD.
Acknowledgements  The authors like to thank Dr. Anna Falk for the
kind gift of the AF22 cells, MSc. Chris Sackmann for the kind gift
of the CD63-GFP SH-SY5Y cells and Dr. Jakob Domert for expert
illustrations. Xandra Breakefield is acknowledged for critical proof-
reading of the manuscript. This research was made possible by fund-
ing from the Swedish Research Council (MH: 523-2013-2735), The
Swedish Alzheimer foundation, The Swedish Brain Foundation, the
Hans-Gabriel and Alice Trolle-Wachtmeister Foundation for Medical
Research, Konung Gustaf V:s och Drottning Victorias Frimurarestif-
telse, Marianne and Marcus Wallenberg Foundation, The Swedish
Fund for Research without Animal Experiments, The Swedish Demen-
tia Foundation, the Linköping University Neurobiology Centre and
the County Council of Östergötland. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Author contributions  MSS and AA designed the experimental
approach, performed all the major biochemical and cellular experi-
ments, analysed and interpreted the data, generated the figures, partici-
pated in the study design, and in writing the manuscript. LC carried
out SEC and DOT blot, prepared the Aβ aggregates and participated
in writing the manuscript. CH prepared brain slices and carried out
immunohistochemistry and participated in writing the manuscript.
ML carried out EM and participated in writing the manuscript. LA
provided antibodies and participated in writing the manuscript. MI
provided brain samples and participated in writing the manuscript. MH
conceived the hypothesis, coordinated, and led the study, participated
in study design, data interpretation and in writing the manuscript.
Compliance with ethical standards 
Conflict of interest  The authors have declared that no conflict of inter-
est exists.
Open Access  This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creat​iveco​
mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
References
1. Agholme L, Lindstrom T, Kagedal K, Marcusson J, Hallbeck M
(2010) An in vitro model for neuroscience: differentiation of SH-
SY5Y cells into cells with morphological and biochemical char-
acteristics of mature neurons. J Alzheimers Dis 20:1069–1082.
https​://doi.org/10.3233/JAD-2010-09136​3
2. Agholme L, Nath S, Domert J, Marcusson J, Kagedal K, Hallbeck
M (2013) Proteasome inhibition induces stress kinase dependent
transport deficits—implications for Alzheimer’s disease. Mol Cell
Neurosci 58C:29–39. https​://doi.org/10.1016/j.mcn.2013.11.001
3. Akrap I, Thavamani A, Nordheim A (2016) Vps4A-mediated
tumor suppression upon exosome modulation? Ann Transl Med
4:180. https​://doi.org/10.21037​/atm.2016.04.18
4. Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T,
Wolozin B, Butovsky O, Kügler S, Ikezu T (2015) Depletion of
microglia and inhibition of exosome synthesis halt tau propaga-
tion. Nat Neurosci. https​://doi.org/10.1038/nn.4132
Acta Neuropathologica (2018) 136:41–56 55
5. Bellingham SA, Guo BB, Coleman BM, Hill AF (2012)
Exosomes: vehicles for the transfer of toxic proteins associated
with neurodegenerative diseases? Front Physiol 3:124. https:​ //doi.
org/10.3389/fphys​.2012.00124​
6. Blockhuys S, Celauro E, Hildesjo C, Feizi A, Stal O, Fierro-Gon-
zalez JC, Wittung-Stafshede P (2017) Defining the human cop-
per proteome and analysis of its expression variation in cancers.
Metallomics 9:112–123. https​://doi.org/10.1039/c6mt0​0202a​
7. Braak H, Braak E (1991) Neuropathological staging of Alzheimer-
related changes. Acta Neuropathol 82:239–259
8. Brettschneider J, Del Tredici K, Lee VM, Trojanowski JQ (2015)
Spreading of pathology in neurodegenerative diseases: a focus
on human studies. Nat Rev Neurosci 16:109–120. https​://doi.
org/10.1038/nrn38​87
9. Bulloj A, Leal MC, Xu H, Castano EM, Morelli L (2010) Insulin-
degrading enzyme sorting in exosomes: a secretory pathway for
a key brain amyloid-beta degrading protease. J Alzheimers Dis
19:79–95. https​://doi.org/10.3233/JAD-2010-1206
10. Cosker KE, Segal RA (2014) Neuronal signaling through endo-
cytosis. Cold Spring Harb Perspect Biol. https​://doi.org/10.1101/
cshpe​rspec​t.a0206​69
11. Daniel JA, Chau N, Abdel-Hamid MK, Hu L, von Kleist L, Whit-
ing A, Krishnan S, Maamary P, Joseph SR, Simpson F et al (2015)
Phenothiazine-derived antipsychotic drugs inhibit dynamin and
clathrin-mediated endocytosis. Traffic 16:635–654. https​://doi.
org/10.1111/tra.12272​
12. Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow
AR, Zhu L, Vanderburg CR, McLean PJ (2012) Exosomal cell-to-
cell transmission of alpha synuclein oligomers. Mol Neurodegener
7:42. https​://doi.org/10.1186/1750-1326-7-42
13. DeKosky ST, Scheff SW (1990) Synapse loss in frontal cortex
biopsies in Alzheimer’s disease: correlation with cognitive sever-
ity. Ann Neurol 27:457–464. https​://doi.org/10.1002/ana.41027​
0502
14. Dinkins MB, Dasgupta S, Wang G, Zhu G, Bieberich E (2014)
Exosome reduction in vivo is associated with lower amyloid
plaque load in the 5XFAD mouse model of Alzheimer’s disease.
Neurobiol Aging 35:1792–1800. https​://doi.org/10.1016/j.neuro​
biola​ging.2014.02.012
15. Domert J, Rao SB, Agholme L, Brorsson AC, Marcusson J,
Hallbeck M, Nath S (2014) Spreading of amyloid-beta pep-
tides via neuritic cell-to-cell transfer is dependent on insufficient
cellular clearance. Neurobiol Dis. https​: //doi.org/10.1016/j.
nbd.2013.12.019
16. Edgar JR, Willen K, Gouras GK, Futter CE (2015) ESCRTs
regulate amyloid precursor protein sorting in multivesicular
bodies and intracellular amyloid-beta accumulation. J Cell Sci
128:2520–2528. https​://doi.org/10.1242/jcs.17023​3
17. Eitan E, Hutchison ER, Marosi K, Comotto J, Mustapic M,
Nigam SM, Suire C, Maharana C, Jicha GA, Liu D et al (2016)
Extracellular vesicle-associated abeta mediates trans-neuronal
bioenergetic and Ca2 + -handling deficits in alzheimer’s disease
models. NPJ Aging Mech Dis. https​://doi.org/10.1038/npjam​
d.2016.19
18. Eitan E, Suire C, Zhang S, Mattson MP (2016) Impact of lyso-
some status on extracellular vesicle content and release. Ageing
Res Rev 32:65–74. https​://doi.org/10.1016/j.arr.2016.05.001
19. Englund H, Sehlin D, Johansson A-SS, Nilsson LN, Geller-
fors P, Paulie S, Lannfelt L, Pettersson FE (2007) Sensitive
ELISA detection of amyloid-beta protofibrils in biological
samples. J Neurochem 103:334–345. https​: //doi.org/10.111
1/j.1471-4159.2007.04759​.x
20. Falk A, Koch P, Kesavan J, Takashima Y, Ladewig J, Alexander
M, Wiskow O, Tailor J, Trotter M, Pollard S et al (2012) Capture
of neuroepithelial-like stem cells from pluripotent stem cells pro-
vides a versatile system for in vitro production of human neurons.
PLoS One 7:e29597. https​://doi.org/10.1371/journ​al.pone.00295​
97
21. Fevrier B, Raposo G (2004) Exosomes: endosomal-derived vesi-
cles shipping extracellular messages. Curr Opin Cell Biol 16:415–
421. https​://doi.org/10.1016/j.ceb.2004.06.003
22. Fiandaca MS, Kapogiannis D, Mapstone M, Boxer A, Eitan E,
Schwartz JB, Abner EL, Petersen RC, Federoff HJ, Miller BL
et al (2015) Identification of preclinical Alzheimer’s disease by a
profile of pathogenic proteins in neurally derived blood exosomes:
a case-control study. Alzheimers Dement 11(600–607):e601. https​
://doi.org/10.1016/j.jalz.2014.06.008
23. Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G,
Dibaj P, Bakhti M, Regen T, Hanisch UK, Simons M (2011)
Selective transfer of exosomes from oligodendrocytes to micro-
glia by macropinocytosis. J Cell Sci 124:447–458. https​://doi.
org/10.1242/jcs.07408​8
24. Fruhbeis C, Frohlich D, Kuo WP, Amphornrat J, Thilemann S,
Saab AS, Kirchhoff F, Mobius W, Goebbels S, Nave KA et al
(2013) Neurotransmitter-triggered transfer of exosomes mediates
oligodendrocyte-neuron communication. PLoS Biol 11:e1001604.
https​://doi.org/10.1371/journ​al.pbio.10016​04
25. Fu H, Hussaini SA, Wegmann S, Profaci C, Daniels JD, Herman
M, Emrani S, Figueroa HY, Hyman BT, Davies P et al (2016) 3D
Visualization of the temporal and spatial spread of tau pathology
reveals extensive sites of tau accumulation associated with neu-
ronal loss and recognition memory deficit in aged tau transgenic
mice. PLoS One. https​://doi.org/10.1371/journ​al.pone.01594​63
26. Goetzl EJ, Boxer A, Schwartz JB, Abner EL, Petersen RC, Miller
BL, Kapogiannis D (2015) Altered lysosomal proteins in neu-
ral-derived plasma exosomes in preclinical Alzheimer disease.
Neurology 85:40–47. https​://doi.org/10.1212/WNL.00000​00000​
00170​2
27. Gouras G, Tampellini D, Takahashi R, Capetillo-Zarate E (2010)
Intraneuronal beta-amyloid accumulation and synapse pathology
in Alzheimer’s disease. Acta Neuropathol 119:523–541. https​://
doi.org/10.1007/s0040​1-010-0679-9
28. Guo JL, Lee VM (2014) Cell-to-cell transmission of pathogenic
proteins in neurodegenerative diseases. Nat Med 20:130–138.
https​://doi.org/10.1038/nm.3457
29. Hamlett ED, Goetzl EJ, Ledreux A, Vasilevko V, Boger HA,
LaRosa A, Clark D, Carroll SL, Carmona-Iragui M, Fortea J et al
(2016) Neuronal exosomes reveal Alzheimer’s disease biomarkers
in Down syndrome. Alzheimers Dement. https:​ //doi.org/10.1016/j.
jalz.2016.08.012
30. Hasegawa T, Konno M, Baba T, Sugeno N, Kikuchi A, Kobayashi
M, Miura E, Tanaka N, Tamai K, Furukawa K et al (2011) The
AAA-ATPase VPS4 regulates extracellular secretion and lysoso-
mal targeting of alpha-synuclein. PLoS One 6:e29460. https:​ //doi.
org/10.1371/journ​al.pone.00294​60
31. Horikoshi Y, Sakaguchi G, Becker AG, Gray AJ, Duff K, Aisen
PS, Yamaguchi H, Maeda M, Kinoshita N, Matsuoka Y (2004)
Development of Abeta terminal end-specific antibodies and sen-
sitive ELISA for Abeta variant. Biochem Biophys Res Commun
319:733–737. https​://doi.org/10.1016/j.bbrc.2004.05.051
32. Hsia AY, Masliah E, McConlogue L, Yu GQ, Tatsuno G, Hu K,
Kholodenko D, Malenka RC, Nicoll RA, Mucke L (1999) Plaque-
independent disruption of neural circuits in Alzheimer’s disease
mouse models. Proc Natl Acad Sci USA 96:3228–3233
33. Joshi P, Turola E, Ruiz A, Bergami A, Libera DD, Benussi L,
Giussani P, Magnani G, Comi G, Legname G et al (2014) Micro-
glia convert aggregated amyloid-β into neurotoxic forms through
the shedding of microvesicles. Cell Death Differ 21:582–593.
https​://doi.org/10.1038/cdd.2013.180
34. Kumari S, Mg S, Mayor S (2010) Endocytosis unplugged: mul-
tiple ways to enter the cell. Cell Res 20:256–275. https​://doi.
org/10.1038/cr.2010.19
13

56
35. Lane RE, Korbie D, Anderson W, Vaidyanathan R, Trau M (2015)
Analysis of exosome purification methods using a model liposome
system and tunable-resistive pulse sensing. Sci Rep 5:7639. https​
://doi.org/10.1038/srep0​7639
36. Logozzi M, De Milito A, Lugini L, Borghi M, Calabro L, Spada
M, Perdicchio M, Marino ML, Federici C, Iessi E et al (2009)
High levels of exosomes expressing CD63 and caveolin-1 in
plasma of melanoma patients. PLoS One 4:e5219. https​://doi.
org/10.1371/journ​al.pone.00052​19
37. Maas SLN, De Vrij J, Broekman MLD (2014) Quantification and
size-profiling of extracellular vesicles using tunable resistive pulse
sensing. Jove-J Vis Exp. https​://doi.org/10.3791/51623​
38. McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ,
Vbeyreuther K, Bush AI, Masters CL (1999) Soluble pool
of Abeta amyloid as a determinant of severity of neurode-
generation in Alzheimer’s disease. Ann Neurol 46:860–866.
https ​ : //doi.org/10.1002/1531-8249(19991 ​ 2 )46:6%3C860​
::AID-ANA8%3E3.0.CO;2-M
39. Narasimhan S, Guo JL, Changolkar L, Stieber A, McBride JD,
Silva LV, He Z, Zhang B, Gathagan RJ, Trojanowski JQ et al
(2017) Pathological tau strains from human brains recapitu-
late the diversity of tauopathies in nontransgenic mouse brain.
J Neurosci 37:11406–11423. https​://doi.org/10.1523/JNEUR​
OSCI.1230-17.2017
40. Nath S, Agholme L, Kurudenkandy FR, Granseth B, Marcus-
son J, Hallbeck M (2012) Spreading of neurodegenerative
pathology via neuron-to-neuron transmission of beta-amyloid.
J Neurosci 32:8767–8777. https ​ : //doi.org/10.1523/JNEUR​
OSCI.0615-12.2012
41. Ngolab J, Trinh I, Rockenstein E, Mante M, Florio J, Trejo M,
Masliah D, Adame A, Masliah E, Rissman RA (2017) Brain-
derived exosomes from dementia with Lewy bodies propagate
α-synuclein pathology. Acta Neuropathol Commun 5:46. https​://
doi.org/10.1186/s4047​8-017-0445-5
42. Perez-Gonzalez R, Gauthier SA, Kumar A, Levy E (2012) The
exosome secretory pathway transports amyloid precursor pro-
tein carboxyl-terminal fragments from the cell into the brain
extracellular space. J Biol Chem 287:43108–43115. https​://doi.
org/10.1074/jbc.M112.40446​7
43. Pigino G, Morfini G, Atagi Y, Deshpande A, Yu C, Jungbauer L,
LaDu M, Busciglio J, Brady S (2009) Disruption of fast axonal
transport is a pathogenic mechanism for intraneuronal amy-
loid beta. Proc Natl Acad Sci USA 106:5907–5912. https​://doi.
org/10.1073/pnas.09012​29106​
44. Polanco J, Li C, Durisic N, Sullivan R, Götz J (2018) Exosomes
taken up by neurons hijack the endosomal pathway to spread to
interconnected neurons. Acta Neuropathol Commun 6:10. https​
://doi.org/10.1186/s4047​8-018-0514-4
45. Raj A, Kuceyeski A, Weiner M (2012) A network diffusion model
of disease progression in dementia. Neuron 73:1204–1215. https​
://doi.org/10.1016/j.neuro​n.2011.12.040
46. Rajendran L, Honsho M, Zahn TR, Keller P, Geiger KD, Verkade
P, Simons K (2006) Alzheimer’s disease beta-amyloid peptides
are released in association with exosomes. Proc Natl Acad Sci
USA 103:11172–11177. https:​ //doi.org/10.1073/pnas.060383​ 8103​
47. Saheki Y, De Camilli P (2012) Synaptic vesicle endocytosis. Cold
Spring Harb Perspect Biol 4:a005645. https​://doi.org/10.1101/
cshpe​rspec​t.a0056​45
48. Salido-Guadarrama I, Romero-Cordoba S, Peralta-Zaragoza
O, Hidalgo-Miranda A, Rodriguez-Dorantes M (2014) Micro-
RNAs transported by exosomes in body fluids as mediators of
13
Acta Neuropathologica (2018) 136:41–56
intercellular communication in cancer. Onco Targets Ther 7:1327–
1338. https​://doi.org/10.2147/OTT.S6156​2
49. Saman S, Kim W, Raya M, Visnick Y, Miro S, Saman S, Jack-
son B, McKee AC, Alvarez VE, Lee NC et al (2012) Exosome-
associated tau is secreted in tauopathy models and is selectively
phosphorylated in cerebrospinal fluid in early Alzheimer dis-
ease. J Biol Chem 287:3842–3849. https​://doi.org/10.1074/jbc.
M111.27706​1
50. Selkoe DJ (2008) Soluble oligomers of the amyloid beta-pro-
tein impair synaptic plasticity and behavior. Behav Brain Res
192:106–113. https​://doi.org/10.1016/j.bbr.2008.02.016
51. Stokin GB, Lillo C, Falzone TL, Brusch RG, Rockenstein E,
Mount SL, Raman R, Davies P, Masliah E, Williams DS et al
(2005) Axonopathy and transport deficits early in the pathogen-
esis of alzheimer’s disease. Science 307:1282–1288. https​://doi.
org/10.1126/scien​ce.11056​81
52. Thal DR, Rub U, Orantes M, Braak H (2002) Phases of a beta-
deposition in the human brain and its relevance for the develop-
ment of AD. Neurology 58:1791–1800. https​://doi.org/10.1212/
WNL.58.12.1791
53. Thal DR, Rub U, Schultz C, Sassin I, Ghebremedhin E, Del
Tredici K, Braak E, Braak H (2000) Sequence of Abeta-protein
deposition in the human medial temporal lobe. J Neuropathol Exp
Neurol 59:733–748. https​://doi.org/10.1093/jnen/59.8.733
54. Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation
and characterization of exosomes from cell culture supernatants
and biological fluids. Curr Protoc Cell Biol 3:22. https​://doi.
org/10.1002/04711​43030​.cb032​2s30
55. Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composi-
tion, biogenesis and function. Nat Rev Immunol 2:569–579. https​
://doi.org/10.1038/nri85​5
56. Tucker S, Moller C, Tegerstedt K, Lord A, Laudon H, Sjodahl
J, Soderberg L, Spens E, Sahlin C, Waara ER et al (2015) The
murine version of BAN2401 (mAb158) selectively reduces amy-
loid-beta protofibrils in brain and cerebrospinal fluid of tg-ArcSwe
mice. J Alzheimers Dis 43:575–588. https:​ //doi.org/10.3233/JAD-
14074​1
57. Tucker S, Möller C, Tegerstedt K, Lord A, Laudon H, Sjödahl
J, Söderberg L, Spens E, Sahlin C, Waara ER et al (2015) The
murine version of BAN2401 (mAb158) selectively reduces
amyloid-β protofibrils in brain and cerebrospinal fluid of tg-Arc-
Swe mice. JAD 43:575–588. https​://doi.org/10.3233/JAD-14074​
1
58. Urbanelli L, Magini A, Buratta S, Brozzi A, Sagini K, Polchi A,
Tancini B, Emiliani C (2013) Signaling pathways in exosomes
biogenesis, secretion and fate. Genes (Basel) 4:152–170. https​://
doi.org/10.3390/genes​40201​52
59. Vella LJ, Hill AF, Cheng L (2016) Focus on extracellular vesi-
cles: exosomes and their role in protein trafficking and biomarker
potential in alzheimer’s and parkinson’s disease. Int J Mol Sci
17:173. https​://doi.org/10.3390/ijms1​70201​73
60. Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe
MS, Rowan MJ, Selkoe DJ (2002) Naturally secreted oligom-
ers of amyloid beta protein potently inhibit hippocampal long-
term potentiation in  vivo. Nature 416:535–539. https​: //doi.
org/10.1038/41653​5a
61. Walsh DM, Selkoe DJ (2004) Deciphering the molecular basis of
memory failure in Alzheimer’s disease. Neuron 44:181–193. https​
://doi.org/10.1016/j.neuro​n.2004.09.010